Chronic or persistent pain is a difficult medical problem which affects millions of people in the U.S. because of no effective treatment. Therefore it is important to understand the mechanisms of persistent pain thereby allowing new treatment paradigms to be developed. An important discovery during the last few decades is that persistent pain is an abnormal state of the nervous system where neurons in the periphery and the central nervous system are sensitized, called as peripheral and central sensitization, respectively. A recent emerging concept in persistent pain is that elevated levels of spinal reactive oxygen species (ROS) play a critical role in central sensitization. The focus of this proposal is the role of ROS in capsaicin induced secondary hyperalgesia due to central sensitization. The overall hypothesis is that increased production of mitochondrial superoxides in neurons in response to excessive nociceptive inputs elevates ROS in the spinal cord. The elevated ROS then activate protein kinases, leading to increased NMDA receptor activation and thus central sensitization. Four specific aims are proposed to test the following hypotheses in the mouse: 1) elevation of spinal ROS is critical for production of capsaicininduced secondary hyperalgesia, thus removing ROS reduces pain; 2) elevation of spinal ROS is due to increased production of superoxides in neurons in response to nociceptive afferent inputs; 3) superoxide dismutase (SOD) is a major mechanism for reducing spinal ROS effects; and 4) elevated spinal ROS will sensitize dorsal horn neurons by activating protein kinases, leading to enhanced phosphorylation of NMDA receptors and thus central sensitization. The present proposal will test these hypotheses using a multidisciplinary approach. Successful completion of these aims will uncover important spinal mechanisms of persistent pain and may lead to the development of new analgesic drugs based on free radical scavengers.